Ayyasamy Vanniarajan

Novel mitochondrial mutation in the ND4 gene associated with Leigh syndrome

We analyzed the complete mitochondrial genome of a 3‐month‐old female child with basal ganglionic lesions and other clinical features suggestive of Leigh syndrome, which is caused by variations in mitochondrial and nuclear genes. Our study revealed a novel, homoplasmic T11984C missense mutation in ND4 gene, which replaces a highly conserved amino acid tyrosine with histidine. Computational analysis showed that this mutation alters the secondary structure of ND4 subunit. As the mutation observed in this study was novel and homoplasmic, we speculate that there could be interplay of this mitochondrial mutation along with nuclear gene(s) in the pathogenesis.

Mitochondrial dysfunction and genetic heterogeneity in chronic periodontitis

We performed an extensive study on mitochondrial dysfunction in chronic periodontitis (CP). Electron microscopic analysis of gingival cells revealed abnormal mitochondria in 60% of the patients. Mitochondrial membrane potential and oxygen consumption of gingival cells were reduced by 4 fold and 5.8 fold, respectively; whereas ROS production was increased by 18%. The genetic analysis by complete mitochondrial DNA sequencing revealed the identification of 14 novel mutations only in periodontal tissues but not in the blood, suggesting a role of oxidative stress on periodontal tissues. Thus, our functional and genetic analysis provided an evidence for the mitochondrial dysfunction in CP.

Clinical and genetic uniqueness in an individual with MELAS.

Mitochondrial encephalopathy lactic acidosis stroke like episodes (MELAS) is a progressive neurodegenerative disorder with varying age of onset. It is a clinically and genetically heterogeneous disease. Molecular etiology of MELAS is not known in several cases. We have identified a unique individual with late onset MELAS at the age of 55 years. We have analyzed the complete mitochondrial genome of the tissue and blood samples of the patient. One novel heteroplasmic mutation (C13565A) in NADH dehydrogenase 5 subunit (ND5) gene was found only in the tissue sample but not in the blood. This mutation is missense causing a change of amino acid serine to tyrosine at position 410. This mutation was found neither in controls nor in world populations. This study has also confirmed ND5 as a hotspot for the mitochondrial diseases. This will be of great help for the clinicians in the diagnosis of MELAS.

Contribution of muscle biopsy and genetics to the diagnosis of chronic progressive external opthalmoplegia of mitochondrial origin

Chronic progressive external opthalmoplegia (CPEO) is the most common phenotypic syndrome of the mitochondrial myopathies. Muscle biopsy, which provides important morphological clues for the diagnosis of mitochondrial disorders, is normal in approximately 25% of patients with CPEO, thus necessitating molecular genetic analysis for more accurate diagnosis. We aimed to study the utility of various histochemical stains in the diagnosis of CPEO on muscle biopsy and to correlate these results with genetic studies. Between May 2005 and November 2007 all 45 patients diagnosed with CPEO were included in the study (23 males; mean age at presentation, 35 years). Thirty-nine patients had CPEO only and six had CPEO plus; two had a positive family history but the remaining 39 patients had sporadic CPEO. Muscle biopsy samples were stained with hematoxylin and eosin, modified Gomoris trichrome stain, succinic dehydrogenase (SDH), cytochrome C oxidase (COX) and combined COX-SDH. Ragged red fibers were seen in 27 biopsies; seven showed characteristics of neurogenic atrophy only, and 11 were normal. The abnormal fibers were best identified on COX-SDH stain. A complete mitochondrial genome was amplified in muscle and blood samples of all patients. Mutations were found in transfer RNA, ribosomal RNA, ND, CYTB, COX I, II and III genes. Mitochondrial gene mutations were found in ten of the 11 patients with a normal muscle biopsy. The genetic mutations were classified according to their significance. The observed muscle biopsy findings were correlated with genetic mutations noted. Histological studies should be combined with genetic studies for the definitive diagnosis of CPEO syndrome.

Mitochondrial DNA variations associated with recurrent pregnancy loss among Indian women.

Several genetic factors have been found to be associated with recurrent pregnancy loss (RPL). However, not many attempts have been made to associate the mitochondrial DNA (mtDNA) variations with RPL. Therefore, we have analyzed the complete mtDNA of 100 women with RPL and 12 aborted fetal tissues. Our analysis revealed a total of 681 variations, most of which were in NADH Dehydrogenase (ND) genes that encode mitochondrial enzyme Complex I. Presence of T4216C variation (ND1 gene) in 9% of the RPL women and several pathogenic, and novel mutations suggest the role of mtDNA variations in RPL.

Haplogroup Heterogeneity of LHON Patients Carrying the m.14484T>C Mutation in India

PURPOSE To investigate the clinical and mitochondrial DNA (mtDNA) haplogroup background of Indian Leber hereditary optic neuropathy (LHON) patients carrying the m.14484T>C mutation. METHODS Detailed clinical investigation and complete mtDNA sequencing analysis was carried out for eight Indian LHON families with the m.14484T>C mutation. Haplogroup was constructed based on the evolutionarily important mtDNA variants. RESULTS In the present study, we characterized eight unrelated probands selected from 187 LHON cases. The overall penetrance of the disease was estimated to be 19.75% (16/81) in eight pedigrees with the m.14484T>C mutation and showed substantially higher sex bias (male: female = 13:3). The mtDNA haplogrouping revealed that they belong to diverse haplogroups; i.e., F1c1, M31a, U2a, M*, I1, M6, M3a1, and R30a. Interestingly, we did not find an association of the m.14484T>C mutation with any specific haplogroup within the Indian population. We also did not find any secondary mutation(s) in these pedigrees, which might affect the clinical expression of LHON. CONCLUSIONS Contrary to earlier reports showing preferential association of the m.14484T>C mutation with western Eurasian haplogroup J and increased clinical penetrance when present in J1 subhaplogroup background, the present study shows that m.14484T>C arose independently in a different mtDNA haplogroup and ethnic background in India, which may influence the clinical expression of the disease.

Lipid storage myopathies with unusual clinical manifestations.

We describe the clinical presentation, course and pathologic findings found in three adult patients with lipid storage myopathy. Excessive lipid storage was found in Type 1 fibers of muscle. Clinical improvement on oral levo-carnitine therapy suggests the possibility of carnitine deficiency as the most likely etiology in two of the patients and one had mitochondrial myopathy confirmed on genetic analysis.

Epilepsia partialis continua in mitochondrial dysfunction: Interesting phenotypic and MRI observations

An 11-year-old girl manifested with photophobia, ptosis, external ophthalmoplegia, hypotonia, weakness of proximal limb muscles, hyporeflexia, and generalized seizures (six months). Her elder sister had had uncontrolled seizures and photophobia and died at seven years of age. In the patient, serum lactate was high (55 mg/dl). Muscle biopsy revealed characteristic ragged red and ragged blue fibers, diagnostic of mitochondrial cytopathy. Sequencing of the complete mitochondrial genome of the DNA obtained from the muscle biopsy of the patient did not show any characteristic mutation. Four months later, the girl was admitted with a one-week history of epilepsia partialis continua (EPC). EEG revealed Periodic Lateralized Epileptiform Discharges (PLEDs), once in 2-4 seconds, over the right temporo-occipital leads. MRI revealed signal change of right motor cortex, which had restricted diffusion. MR spectroscopy (MRS) from this region revealed lactate peak. EPC remained refractory to multiple anti-epileptic drugs, immuno-modulators, coenzyme-Q, and carnitine. This thought provoking report expands the spectrum of mitochondrial cytopathies.

Author Response: Penetrance of the LHON Mutation m.11778G>A May Depend on Factors Other Than Haplotype or Heteroplasmy Rate

We appreciate the effort shown by the authors and their comments on our article ‘‘Leber’s Hereditary Optic Neuropathy-Specific Mutation m.11778G>A Exists on Diverse Mitochondrial Haplogroups in India.’’ Leber’s hereditary optic neuropathy (LHON) is the most common, well-diagnosed, and maternally inherited mitochondrial disease. In this case, we would emphasize the statement that LHON is one of the most well characterized maternally inherited mitochondrial disease. We do agree with the statement that penetrance may depend on the age of individuals who carry the mutation. To date, our follow-up data do not reveal nonsymptomatic mutation carriers to have developed visual impairment and LHON. The data presented are to the best of our knowledge, till the time the manuscript was written. However, most of the families are in contact with the clinicians and have been informed about their genetic diagnosis. The families were followed up for the period of study (2005–2016), and patients were included if they developed visual disturbance. As discussed in the article, 13 out of 64 LHON families with m.11778G>A mutation were observed in heteroplasmic condition, and the remaining 51 were homoplasmic. In total (22 out of 145), 15% of manifesting mutation carriers was present with heteroplasmic form, while 31% of mutation carriers (124 out of 398) were present with homoplasmic condition. We do agree with the authors that knowledge of environmental factors (tobacco smoke) would greatly help in answering the penetrance of these LHON pedigrees. However, we realized during the study that quite a significant percentage of participants were not ready to share the details about their smoking and alcohol consumption history in an accurate way. At present, we lack proper details of the tobacco smoke and alcohol consumption for all the pedigrees and the amount of information, which we have, is not sufficient to make any specific conclusion about this issue. Keeping this in mind, we have decided to exclude this information from this study. During the initial analysis, 41 out of 64 index cases had positive family history of LHON. Seven affected individuals have mothers clinically manifesting LHON. During the course of this study, all the index cases were investigated for multiorgan involvement. We observed one patient from the index case had mild hepatosplenomegaly, while the other patients from index cases do not have any organ involvement. Hence, we would not be able to make conclusions about the multiorgan involvement and heteroplasmy level. None of the tRNA variants scored >11 on the Yarham score. One of the limitations was that we do not have all the information required for predicting the Yarham score for each patient due to absence of histology and transmitochondrial cybrid study for all the mutations. During initial clinical diagnosis of LHON-like symptoms for the patients, blood samples were collected for genetic diagnosis, which was confirmed by the presence of mutation. We, therefore, do not agree with collection of invasive samples like muscle biopsy in all the cases, but it was collected for a few cases where patients gave their consent. Hence, we were not able to perform all the analysis for predicting the Yarham score for additional variants observed together with m.11778G>A mutation. We also think it would be very difficult to segregate the effect of additional variant, since patients already have pathogenic mutation. We do agree with authors that pathogenicity of variants may be confirmed by using single fiber and cybrid analysis. However, it is not possible for us to make cybrids for all the variants; nevertheless, for the continuation of this project, we have a plan to select a few of the potential variants to study their role in different haplogroup backgrounds.